本文選題：食管鱗狀細(xì)胞癌 + miR-153-5p��； 參考：《南京醫(yī)科大學(xué)》2017年博士論文

【摘要】：背景和目的:食管癌是常見的惡性腫瘤,發(fā)病率和病死率在中國的惡性腫瘤中位列第五和第四。中國每年發(fā)病病例占全球食管癌一半以上,主要組織學(xué)類型是食管鱗狀細(xì)胞癌。盡管手術(shù)、放療和化療等針對于食管癌的多學(xué)科綜合治療不斷進(jìn)步,但是食管癌患者的預(yù)后仍然不樂觀。因此,進(jìn)一步認(rèn)識該疾病的分子特征,推進(jìn)臨床生物標(biāo)志物應(yīng)用以及改善治療模式有廣泛的社會需求和重要的臨床義。微小RNA(microRNA,miRNA)是一類內(nèi)源性短鏈(長度約約22個核苷酸)的非編碼單鏈RNA,主要通過綁定3'非翻譯區(qū)(UTR)調(diào)節(jié)目標(biāo)基因的表達(dá),導(dǎo)致翻譯抑制。越來越多的證據(jù)表明miRNA可以調(diào)節(jié)腫瘤發(fā)生、發(fā)展影響腫瘤細(xì)胞浸潤和轉(zhuǎn)移,miRNAs的致癌或抑癌的功效取決于miRNA作用的靶基因。近年來,miR-153-5p被發(fā)現(xiàn)是一種人類癌癥的抑癌因子,調(diào)節(jié)腫瘤抑癌基因、參與腫瘤的生長、轉(zhuǎn)移和侵潤,miR-153-5p在ESCC的作用機(jī)制仍不清楚。我們在miRDB檢索miR-153-5p直接靶向因子,其中將WT1(腎母細(xì)胞瘤抑癌基因1)作為研究對象。WT1位于人染色體11P13,有報道在多種惡性腫瘤中發(fā)現(xiàn)WT1起抑癌因子作用。因此,本研究旨在探索miR153-5p在ESCC中的作用,評估臨床資料,探討其在食管鱗狀細(xì)胞癌中的生物功能。探索ESCC細(xì)胞中miR-153-5p如何調(diào)控WT1表達(dá)。本課題包括以下三部分:第一部分:食管鱗狀細(xì)胞癌組織中異常表達(dá)miRNA篩選及血清miRNA-153-5p與臨床病理特征相關(guān)性分析;第二部分:體外調(diào)控miRNA-153-5p表達(dá)對食管癌細(xì)胞系TE-1增殖、侵襲的影響;第三部分:miRNA-153-5p靶基因的初步研究。第一部分食管鱗狀細(xì)胞癌組織中異常表達(dá)miRNA篩選及血清miRNA-153-5p與臨床病理特征相關(guān)性分析方法:1.收集標(biāo)本,包括47例食管鱗狀上皮細(xì)胞癌患者和50名健康人選血清標(biāo)本,以及上述47例食管鱗狀上皮細(xì)胞癌組織與其相應(yīng)配對的癌旁正常組織標(biāo)本。2.隨機(jī)抽取4例食管癌病例和4例健康人選的血清,運(yùn)用Agilent miRNA芯片檢測miRNA表達(dá)情況,并對差異表達(dá)基因進(jìn)行初步功能分析,篩選出顯著異常表達(dá)的十二個miRNA;同樣在這4例食管鱗狀上皮細(xì)胞癌組織與其相應(yīng)配對的癌旁組織標(biāo)本進(jìn)行對照分析。3.進(jìn)一步運(yùn)用qRT-PCR檢測上述12種miRNA中的7種miRNA表達(dá)情況水平,包括5個食管癌中表達(dá)上調(diào)的miRNA(miR-25、miR-155、miR-214、miR-200c、miR-92-c)和2個食管癌中表達(dá)下調(diào)的miRNA基因(miR-518b 和 miR-153-5p)。4.在50例健康人選和47例ESCC病例依據(jù)血清miRNA-153-5p表達(dá)水平,運(yùn)用ROC曲線分析其與食管鱗狀細(xì)胞癌病人血清miRNA-153-5p表達(dá)水平與健康人群血清表達(dá)水平以及與食管鱗狀細(xì)胞癌患者淋巴結(jié)轉(zhuǎn)移、腫瘤長度、腫瘤分期、腫瘤分化程度等之間的相關(guān)性。5.統(tǒng)計學(xué)分析:采用統(tǒng)計軟件SPSS 22.0對實(shí)驗(yàn)數(shù)據(jù)進(jìn)行統(tǒng)計分析。血清miRNA-153-5p表達(dá)與臨床診斷相關(guān)性分析進(jìn)行聚類分析,ROC曲線,所有結(jié)果均以means ± SE表示,student t-test進(jìn)行數(shù)據(jù)比較分析,P0.05為有統(tǒng)計學(xué)意義。結(jié)果:1.Agilent miRNA芯片檢測分析得到研究發(fā)現(xiàn)有25個miRNA表達(dá)水平存在顯著差異(P0.05和差異倍數(shù)≥2),包括13個高表達(dá)miRNA和12個低表達(dá)miRNA。組織miRNA表達(dá)水平與血清的研究結(jié)果一致。2.已被基因芯片檢測的七個血清miRNA包括miR-25、miR-518b、miR-155、miR-214、miR-200c、miR-92-c 和 miR-153-5p,被 qRT-PCR 再次檢測,總體qRT-PCR表達(dá)趨勢與芯片結(jié)果一致;7個miRNA在兩種組織樣本qRT-PCR的表達(dá)趨勢與血清結(jié)果一至。選擇差異最為顯著的具有負(fù)向調(diào)控作用的miRNA-153-5p作為研究對象。3.與健康人相比ESCC患者的血清miR-153-5p表達(dá)水平較低,ESCC患者的血清miR-153-5p表達(dá)水平與食管鱗狀細(xì)胞癌患者的淋巴結(jié)轉(zhuǎn)移、腫瘤長度和腫瘤TNM分期相關(guān),與腫瘤分化程度無顯著相關(guān)性。結(jié)論:1.本研究在食管癌組織中一共篩選分析得到25個差異表達(dá)的miRNA,其中有13個為上調(diào)miRNA基因,12個為下調(diào)miRNA基因。2.miR-153-5p在食管鱗狀細(xì)胞癌患者血清和食管鱗癌組織中顯著低表達(dá),選擇差異最為顯著的具有負(fù)向調(diào)控作用的miRNA-153-5p作為食管鱗狀細(xì)胞癌研究對象。3.食管鱗狀細(xì)胞癌患者血清中miR-153-5p的表達(dá)水平在診斷食管鱗狀細(xì)胞癌有潛在價值;與食管鱗狀細(xì)胞癌患者的淋巴結(jié)轉(zhuǎn)移、腫瘤長度和腫瘤TNM分期相關(guān),與腫瘤分化程度無顯著相關(guān)性,在評估食管鱗狀細(xì)胞癌的臨床特征具有良好的潛在應(yīng)用價值。第二部分體外miR-153-5p表達(dá)對食管癌細(xì)胞系生物學(xué)行為的影響方法:1.運(yùn)用 qRT-PCR 檢測 HEEC、EC190、EC9706、SKGT-5 和 TE-1 等細(xì)胞系中miR-153-5p表達(dá)。2.實(shí)驗(yàn)分組為 Control 陰性組、miRNA-153-5pNC 組和 miRNA-153-5p mimics組,利用LipofectamineTM2000轉(zhuǎn)染各組后TE-1細(xì)胞中miR-153-5p mRNA表達(dá)水平檢測。3.MTT增殖實(shí)驗(yàn)檢測各組TE-1細(xì)胞增殖能力影響。4.Transwell侵襲實(shí)驗(yàn)法檢測各組TE-1細(xì)胞的侵襲轉(zhuǎn)移能力。5.Western blot和qRT-PCR檢測各實(shí)驗(yàn)組細(xì)胞中與腫瘤增殖和侵襲相關(guān)蛋白Vimentin和E-cadherin水平表達(dá)。6.統(tǒng)計學(xué)分析:采用SPSS20.0分析數(shù)據(jù),各實(shí)驗(yàn)組獨(dú)立實(shí)驗(yàn)計量資料據(jù)采集使用均數(shù)±標(biāo)準(zhǔn)差,組間均數(shù)比較采用非配對雙側(cè)t檢驗(yàn)。qRT-PCR結(jié)果及腫瘤質(zhì)量組間比較采用方差分析。P0.05為有統(tǒng)計學(xué)意義。結(jié)果:1.在HEEC(正常食管上皮細(xì)胞)miR-153-5p的表達(dá)明顯高于人類食管癌細(xì)胞系 EC190、EC9706、SKGT-5 和 TE-1 等(P0.01),對比對照組,表達(dá)水平顯著上調(diào)(P0.01)。2.MiR-153-5p mimics和相應(yīng)的負(fù)控制成功轉(zhuǎn)染到TE-1細(xì)胞,與NC組織相比,miR-153-5p水平在TE-1細(xì)胞中水平顯著提高(P0.01)。3.MTT測定miR-153-5p過度表達(dá)抑制了 TE-1細(xì)胞的增殖(P0.01)4.Transwell侵襲試驗(yàn)表明,在miR-153-5p mimics組,TE-1細(xì)胞侵襲明顯被抑制(P0.01)。miR-153-5pmimics組細(xì)胞的生長在轉(zhuǎn)染2d后出現(xiàn)顯著抑制(P0.05),并且隨時間的延長而日益顯著。5.Westernblot、qRT-PCR顯示擴(kuò)散和侵襲相關(guān)蛋白表達(dá)改變:與腫瘤發(fā)生、轉(zhuǎn)移密切相關(guān)的vimentin表達(dá)減弱,與維持正常細(xì)胞結(jié)構(gòu)、功能相關(guān)的E-cadherin表達(dá)增強(qiáng)。結(jié)論:1.miR-153-5p在體外食管癌細(xì)胞中水平顯著降低。2.上調(diào)食管癌TE-1細(xì)胞中miR-153-5p表達(dá)可有效抑制食管癌TE-1細(xì)胞的增殖和侵襲能力,并且影響食管癌擴(kuò)散和侵襲相關(guān)蛋白vimentin、E-cadherin 的表達(dá)。第三部分miRNA-153-5p靶基因的初步研究方法:1.開放應(yīng)用程序miRDB、miRBase、starBase和TargetScan等用來分析潛在miR-153-5p的靶向目標(biāo)。2.采用Western blot、qRT-PCR方法用來檢測ESCC組織樣本中WT1和miR-153-5p mRNA水平并建立相關(guān)性。3.Western blot 和 qRT-PCR 檢測 WT1 在 miR-153-5p mimics 組、miR-NC組、對照組中的表達(dá)。4.構(gòu)建野生型重組載體pmirGLO-WT1(WT),通過定向位點(diǎn)突變獲得綁定位點(diǎn)的突變體結(jié)構(gòu)(MUT)。采用雙熒光素酶報告實(shí)驗(yàn)驗(yàn)證miR-153-5p的靶基因。結(jié)果:1.通過生物信息學(xué)分析推測WT1為miR-153-5p靶基因,miR-153-5p的互補(bǔ)序列被預(yù)測在WT1mRNA的3'-UTR。2.ESCC組織樣本中miR-153-5p水平在ESCC組織中低于相應(yīng)的正常組織,WT1mRNA表達(dá)明顯增加,與miR-153-5p水平負(fù)相關(guān)(P0.01)。3.WT1在miR-153-5p mimics組、miR-NC組、對照組中的表達(dá)結(jié)果是與其他兩組相比,miR-153-5p mimics組WT1蛋白表達(dá)和WT1 mRNA水平較低(P0.01)。4.食管鱗狀癌細(xì)胞TE-1中,共轉(zhuǎn)染miR-153-5pmimics和野生型3'UTR區(qū)的pmirGLO-WT1重組載體(WT)時,WT組中WT1熒光素酶活性被miR-153-5pmimics抑制,提示miR-153-5p可以通過作用于WT1的3'UTR區(qū)負(fù)向調(diào)控其表達(dá),但在MUT中并沒有(P0.01)。結(jié)論:1.WT1預(yù)測為miR-153-5p的靶基因,2.在食管鱗狀細(xì)胞癌組織中WT1 mRNA表達(dá)水平明顯增加,與miR-153-5p表達(dá)水平呈負(fù)相關(guān)。3.在TE-1細(xì)胞系中,miR-153-5pmimics作用于WT1,并抑制了 WT1的表達(dá)。4.MiR-153-5p通過作用于靶基因WT1的3'UTR,抑制WT1的表達(dá)參與食管癌TE-1細(xì)胞生長調(diào)控。
[Abstract]:Background and purpose: esophageal cancer is a common malignant tumor. The incidence and fatality rate are fifth and fourth in China's malignant tumors. The incidence of cancer in China is more than half of the global esophageal cancer in China every year. The main histological type is squamous cell carcinoma of the esophagus. Although surgery, radiotherapy and chemical therapy are not used for the multidisciplinary treatment of esophageal cancer However, the prognosis of patients with esophageal cancer is still not optimistic. Therefore, further understanding of the molecular characteristics of the disease, the application of clinical biomarkers and the improvement of treatment patterns have extensive social needs and important clinical meanings. Small RNA (microRNA, miRNA) is a class of non coded endogenetic short chains (about 22 nucleotides in length). Chain RNA, which regulates the expression of target genes by binding 3'untranslated region (UTR), leads to translation inhibition. More and more evidence suggests that miRNA can regulate the occurrence of tumor and the development of tumor cell infiltration and metastasis. The effect of miRNAs on carcinogenesis or tumor suppressor depends on the target gene of miRNA. In recent years, miR-153-5p has been found to be a kind of human being. Cancer suppressor factor, regulating tumor suppressor gene, participating in tumor growth, metastasis and invasion, the mechanism of miR-153-5p's action in ESCC is still unclear. We retrieved the direct target factor of miR-153-5p in miRDB, in which WT1 (nephroblastoma tumor suppressor gene 1) was used as the research object.WT1 in the human chromosome 11P13, and there were reports in a variety of malignant tumors. The purpose of this study is to explore the role of WT1 as a tumor suppressor factor. Therefore, the purpose of this study is to explore the role of miR153-5p in ESCC, to evaluate the clinical data and to explore its biological functions in the squamous cell carcinoma of the esophagus. To explore how miR-153-5p regulates the expression of WT1 in ESCC cells. This topic includes the following three parts: Part 1: abnormal expression in the tissues of squamous cell carcinoma of the esophagus MiRNA screening and the correlation analysis of serum miRNA-153-5p and clinicopathological features; the second part: the effect of miRNA-153-5p expression on the proliferation and invasion of the esophageal cancer cell line TE-1 in vitro; the third part: the preliminary study of the miRNA-153-5p target gene. The first part of the esophageal squamous cell carcinoma tissue abnormal expression of miRNA and the serum miRNA-153-5p and miRNA-153-5p The method of correlation analysis of clinicopathological features: 1. collection of specimens, including 47 cases of squamous cell carcinoma of the esophagus and 50 healthy persons, and 47 cases of squamous cell carcinoma of the esophagus and the corresponding normal tissue specimens of the paracancerous tissue adjacent to.2., the serum of 4 cases of tube cancer and 4 healthy persons were randomly selected, and Agi was used. Lent miRNA chips were used to detect the expression of miRNA, and a preliminary functional analysis of differentially expressed genes was used to screen out twelve miRNA with significant abnormal expression. The same 4 cases of esophageal squamous cell carcinoma and corresponding paired para cancerous tissue specimens were compared and analyzed by.3., and qRT-PCR was used to detect 7 mi of the 12 kinds of miRNA. The level of RNA expression, including the up regulated miRNA (miR-25, miR-155, miR-214, miR-200c, miR-92-c) and the down regulated miRNA gene (miR-518b and miR-153-5p) in 2 esophageal cancers in 5 esophageal cancers (miR-518b and miR-153-5p).4. in 50 healthy persons and 47 cases of ESCC cases on the basis of serum levels, and using the curve to analyze the squamous cells of the esophagus The correlation of serum miRNA-153-5p expression level with serum expression level in healthy people and the correlation of.5. with lymph node metastasis, tumor length, tumor stage and tumor differentiation in patients with squamous cell carcinoma of esophagus: statistical analysis of the experimental data by statistical software SPSS 22. The expression of serum miRNA-153-5p and the presence of miRNA-153-5p The correlation analysis of bed diagnosis was analyzed by clustering analysis, ROC curve, all the results were expressed in means + SE, student t-test was compared and analyzed, and P0.05 was statistically significant. The results of 1.Agilent miRNA chip detection analysis showed that there were 25 miRNA expression levels (P0.05 and difference multiple more than 2), including 13 high levels. The expression level of the expression of miRNA and 12 low expression miRNA. tissues was consistent with the results of the study in the serum. The seven serum miRNA, including miR-25, miR-518b, miR-155, miR-214, miR-200c, miR-92-c and miR-153-5p, were detected by the gene chip, and the overall expression trend was consistent with the results of the chip; 7 groups were in two groups. The expression trend of qRT-PCR and the result of sera were 1. The most significant difference was the negative regulation of miRNA-153-5p as the object of study. The level of miR-153-5p expression in the serum of ESCC patients was lower than that of the healthy people, and the level of miR-153-5p expression in the serum of ESCC patients and the lymph node metastasis of patients with squamous cell carcinoma of the tube was swollen. The length of the tumor was correlated with the TNM staging of the tumor, and there was no significant correlation with the degree of tumor differentiation. Conclusion: 1. a total of 25 differentially expressed miRNA were screened and analyzed in the esophageal carcinoma tissue, of which 13 were up to up miRNA gene, and 12 for miRNA gene.2.miR-153-5p in the sera and esophageal squamous cell carcinoma tissues of the patients with esophageal squamous cell carcinoma. The expression level of miRNA-153-5p, which has the most significant difference in the negative regulation, is the target of esophageal squamous cell carcinoma. The expression level of miR-153-5p in the sera of.3. esophageal squamous cell carcinoma is of potential value in the diagnosis of squamous cell carcinoma of the esophagus; the lymph node metastasis, the length of tumor and the T of the tumor in the patients with esophageal squamous cell carcinoma NM staging correlation has no significant correlation with the degree of tumor differentiation, and has good potential application value in evaluating the clinical characteristics of esophageal squamous cell carcinoma. Second the effect of miR-153-5p expression on the biological behavior of esophageal cancer cell lines in vitro: 1. using qRT-PCR to detect miR-15 in HEEC, EC190, EC9706, SKGT-5 and TE-1 3-5p expression of.2. was divided into Control negative group, miRNA-153-5pNC group and miRNA-153-5p mimics group, miR-153-5p mRNA expression in TE-1 cells was detected by LipofectamineTM2000 transfection, and.3.MTT proliferation test was used to detect the effect of.3.MTT proliferation test on the proliferation of TE-1 cells. .5.Western blot and qRT-PCR were used to detect the expression of.6. in the cells with tumor proliferation and invasion related protein Vimentin and E-cadherin in the experimental groups. The SPSS20.0 analysis was used to analyze the data of the experimental groups. The independent experimental data of the experimental groups were collected using the mean standard deviation, and the average number between the groups was compared with the non paired bilateral t test.QRT-PCR. The results and the contrast analysis of.P0.05 were statistically significant. Results: 1. the expression of miR-153-5p in HEEC (normal esophageal epithelial cells) was significantly higher than that of human esophageal carcinoma cell line EC190, EC9706, SKGT-5 and TE-1 (P0.01). Compared with the control group, the expression level was significantly up (P0.01).2.MiR-153-5p mimics and the corresponding negative. The control was successfully transfected into TE-1 cells. Compared with NC tissue, the level of miR-153-5p in TE-1 cells increased significantly (P0.01).3.MTT determination of miR-153-5p overexpression inhibited the proliferation of TE-1 cells (P0.01) 4.Transwell invasion test showed that the invasion of cells in miR-153-5p mimics group was obviously inhibited. The growth was significantly inhibited after transfection of 2D (P0.05), and the expression of proliferation and invasion related protein expression changed with the prolongation of time. QRT-PCR showed changes in the expression of diffusion and invasion related proteins: the expression of vimentin, which was closely related to tumor occurrence and metastasis, was weakened, and the expression of E-cadherin was enhanced with the maintenance of normal cell structure and function related. Conclusion: 1.miR-153-5p in 1.miR-153-5p is enhanced. The level of.2. in esophageal cancer cells significantly reduces the expression of miR-153-5p in esophageal cancer TE-1 cells, which can effectively inhibit the proliferation and invasion of esophageal cancer TE-1 cells, and affect the expression of vimentin and E-cadherin in the proliferation and invasion related proteins of esophageal cancer. The preliminary study method of the third part of the miRNA-153-5p target gene: 1. open application Sequential miRDB, miRBase, starBase, and TargetScan are used to analyze potential miR-153-5p target target.2. using Western blot. QRT-PCR method is used to detect WT1 and miR-153-5p levels in ESCC organization samples. A wild type recombinant vector pmirGLO-WT1 (WT) was constructed to obtain the mutant structure (MUT) of the binding site by directional site mutation. Double luciferase reporter experiment was used to verify the target gene of miR-153-5p. Results: 1. by bioinformatics analysis, WT1 was a miR-153-5p target gene, and the complementary sequence of miR-153-5p was predicted in the 3'-UTR.2.ESCC group of WT1mRNA. The miR-153-5p level in the ESCC tissue was lower than that of the corresponding normal tissue, and the expression of WT1mRNA was significantly increased, and the negative correlation with the miR-153-5p level (P0.01).3.WT1 in the miR-153-5p mimics group, miR-NC group and the control group was compared with the other two groups, and the WT1 protein expression and the lower level of miR-153-5p mimics group were lower than those of the other groups. In the squamous cell carcinoma cell TE-1, when the pmirGLO-WT1 recombinant vector (WT) was co transfected with miR-153-5pmimics and wild type 3'UTR region, the activity of WT1 luciferase in group WT was inhibited by miR-153-5pmimics, suggesting that miR-153-5p could regulate its expression by negative direction acting on 3'UTR region of WT1. 2. in esophageal squamous cell carcinoma, the expression of WT1 mRNA increased significantly, and the expression level of miR-153-5p was negatively correlated with the expression level of.3. in the TE-1 cell line. MiR-153-5pmimics acted on WT1 and inhibited the expression of.4.MiR-153-5p through the 3'UTR of the target gene WT1, and inhibited the expression of WT1 to participate in the regulation of the growth of esophageal cancer cells.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.1

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本文編號：1810508